Deubiquitinase USP19 modulates apoptotic calcium release and endoplasmic reticulum stress by deubiquitinating BAG6 in triple negative breast cancer

Abstract Background Triple‐negative breast cancer (TNBC), a heterogeneous subtype of breast cancer (BC), had poor prognosis. Endoplasmic reticulum (ER) stress was responsible for cellular processes and played a crucial role in the cell function. ER stress is a complex and dynamic process that can induce abnormal apoptosis and death. However, the underlying mechanism of ER stress involved in TNBC is not well defined. Methods We identified ubiquitin‐specific protease 19 (USP19) as a TNBC negative regulator for further investigation. The effects of USP19 on BC proliferation were assessed in vitro using proliferation test and cell‐cycle assays, while the effects in vivo were examined using a mouse tumorigenicity model. Through in vitro flow cytometric analyses and in vivo TUNEL assays, cell apoptosis was assessed. Proteomics was used to examine the proteins that interact with USP19. Results Multiple in vitro and in vivo tests showed that USP19 decreases TNBC cell growth while increasing apoptosis. Then, we demonstrated that USP19 interacts with deubiquitinates and subsequently stabilises family molecular chaperone regulator 6 (BAG6). BAG6 can boost B‐cell lymphoma 2 (BCL2) ubiquitination and degradation, thereby raising ER calcium (Ca2+) levels and causing ER stress. We also found that the N 6‐methyladenosine (m6A) “writer” methyltransferase‐like 14 (METTL14) increased global m6A modification. Conclusions Our study reveals that USP19 elevates the intracellular Ca2+ concentration to alter ER stress via regulation of BAG6 and BCL2 stability and may be a viable therapeutic target for TNBC therapy.

methyltransferase-like 14 (METTL14) increased global m 6 A modification.It was the important mechanisms by which USP19 inhibited breast cancer carcinogenesis.

INTRODUCTION
Breast cancer (BC) is the most often diagnosed cancer in the world. 1 TNBC, a heterogeneous subtype of BC, lacks expression of progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2. [2][3][4] Despite the fact that TNBC accounts for 15%−20% of BC incidences, it was responsible for 25% of fatalities.Therapeutic interventions for TNBC are restricted, due to the absence of targetable receptors that differentiate other subtypes.Individuals with TNBC had poor prognosis and overall survival because of higher recurrence rate (over 30%) and shorter survival after metastatic recurrence. 5As a result, understanding the molecular and cellular processes involved in TNBC tumorigenesis is critical.Ubiquitin-specific protease 19 (USP19) is a ubiquitinspecific protease localised in the endoplasmic reticulum (ER), which participates in the regulation of unfolded protein response (UPR). 6,7Through interacting with HSP90 via the CS/p23 domains, USP19 may promote HSP90 folding by giving it the ability to function as an independent partner. 8Jin et al. 9 found that USP19 stabilises Beclin-1 to promote autophagy, by removing the K11linked ubiquitin chain from Beclin-1 at lysine 437, while reduces type I IFN signalling by blocking the interactions between RIG-I-MAVS.Cao-Qi Lei al. 10 found that C607S, an enzyme-inactive mutant site of USP19, failed to inhibit TNFA and IL-1β-triggered NF-κB activation after mutation.][13] ER is an important organelle responsible for protein processing, modification and folding, and plays a crucial role in cell Apoptosis. 146][17] It was generally believed that ER stress sensors activate three signalling pathways: Protein Kinase RNA-like ER Kinase (PERK), Inositol-Required Enzyme 1 (IRE1), and Activating Transcription Factor 6 (ATF6). 18Activated IRE1 19 splices X-Box-Binding Protein 1 (XBP1) mRNA, which can create a powerful transcription factor. Logue 22 reported that inhibition of IRE1 RNase, an ER stress sensor associated with ER stress resolution, can enhance paclitaxel-mediated tumour suppression, and postpone tumour recurrence after treatment for TNBC. Shapiro 24 showed that the anticipatory UPR pathway can be pre-activated by pep-tide hormones, mitogenic steroids, and other effectors, which afterwards cause cytotoxicity in estrogen receptorpositive BC.Here, it was shown that ER stress and UPR are inhibited in TNBC, and play an important role in the tumorigenicity and development of this subtype of BC.
This study revealed that USP19 was expressed in TNBC tissues at low levels.We also found that USP19 overexpression inhibits cell proliferation, induces apoptosis, and regulates cell-cycle assay in TNBC cells.In terms of the underlying mechanism, we identified that USP19 functions as a deubiquitinase that stabilises BAG6 (BAG family molecular chaperone regulator 6, a major histocompatibility complex (MHC) 25 that facilitates tailanchored protein entrance into the ER).BAG6 could serve as a ubiquitinase that poromoted B-cell lymphoma-2 (BCL2) degrade.This study found a significant increase in m 6 A modification level in mRNA of TNBC tissues.Overexpression of METTL14 significantly decreased the expression level and half-life of USP19 mRNA.The METTL14/USP19/BAG6/BCL2 axis increases ER stress and apoptosis in TNBC cells through influencing ER calcium channels.Our research identifies a plausible mechanism for TNBC development and offers a promising method for the treatment.

Tissue samples
RNA sequencing files and routine clinical data of patients diagnosed with BC were collected from The Cancer Genome Atlas (TCGA) data (https://portal.gdc.cancer.gov/)and the Genotype-Tissue Expression (GTEx) database.Then, we assembled dataset containing 1 401 samples and RNA-seq data (Table S2) of 196 patients (Table S3) underwent modified radical mastectomy at the Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, China.And the other deubiquitinating enzyme (DUB) genes annotation files were derived from RNA-seq data from experiments.Tissues were stored in liquid nitrogen for extraction of RNA and protein.

RNA extraction and quantitative qRT-PCR assays
RNA Extraction Kit (Vazyme) was used to extract pure RNA.We used reverse transcription system (Toyobo) to synthesise complementary DNA.qRT-PCR was performed on an Applied Biosystems 7900 Fast Real-Time PCR System using SYBR Green PCR master mix.We normalised gene expression levels against internal control and displayed relative gene expression levels using the 2 −ΔΔCT method.(Table S1)

Fluorescence microscopy
On a confocal dish, cells were cultivated and immediately observed.Cells were cultured, fixed with 4% paraformaldehyde, washed with PBS, then stained with certain antibodies for immunofluorescence microscopy analysis.A Leica DMI3000 B confocal microscope with an oil-immersion objective set at 100 (NA 1.4) was used to evaluate colocalisation images.

Cell proliferation, colony formation and 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay
Cell proliferation ability was measured using EdU assay kit (RiboBio, China).Treated cells were seeded into 24-well plates (2 × 10 4 cells/well) overnight.Cells with EdU reagent were then incubated at 37 • C for 2 h according to the manufacturer's instructions, and after fixation, membrane rupture and Hoechest33342 (400 μl) were added for immunostaining.The green and red fluorescence signals were all observed in the 500−530 and 553−618 nm spectral ranges, respectively.Proliferation was analysed by observing the average number of cells in the three regions of each sample using a Leica microscope.

Fluorescence detection and imaging
Cells were treated for 30 min at 37

Flow cytometric
Apoptosis and cell-cycle were analysed through flow cytometric (FCM) methods.Transfected cells were washed twice with PBS, then trypsinised and centrifuged at 1 200 rpm for 5 min, fixed with 75% ethanol, and stored overnight at −20 • C. Cells were incubated with PI and Annexin V-FITC according to the instructions.All antibody-labelled cells were assayed using a FACSVerse 8 flow cytometer (BD Biosciences), and data were analysed by FlowJo software (Version 10.6.1).Fluo-4 AM (Beyotime) was used to assess intracellular Ca 2+ levels.The treated cells were cultured with a solution of Fluo-4 AM (2 umol/L) at 37 • C for 30 min before being evaluated by flow cytometry.FlowJo software was used to examine the data.

In vitro deubiquitination assays
First, endogenous BAG6 was immunoprecipitated using anti-BAG6 antibody, followed by immunoblotting using ubiquitin antibody to assess endogenous BAG6 ubiquitination.HA-K11 and HA-K29 and Flag-BAG6 were then co-transfected into HEK-293T cells.MG132 treated cells to increase levels of ubiquitinated BAG6.Subsequently, ubiquitinated BAG6 protein was immunoprecipitated using anti-BAG6, anti-Flag antibodies and protein G beads for enrichment.The magnetic beads were boiled mixed with 1× SDS loading buffer and analysed by Western immunoblot analysis.

GST pull-down assay
After being extracted from E. coli BL21, the pure GST-USP19 protein was incubated with either the GST-BAG6 or GST-BCL2 proteins.Protein G beads were used to cleanly separate the GST proteins.Following thorough washing of the beads, immunoblotting analysis was used to identify the bound USP19, BAG6 and BCL2.

Animal experiments
At 4 weeks old, 60 female BALB/c nude mice were divided into four groups at random (n = 6 per group).The flanks of the naked mice received bilateral subcutaneous injections of the stably transfected cell lines MDA-MB-231 and BT-549.Vernier calipers were used to measure the bidimensional tumours every 5 days, and after 4 weeks, the mice were euthanised.Using the equation volume = (width 2 length)/2, the volume of the implanted tumor was estimated.The ethical approval to perform animal experiments is 2019SR512 (December 10, 2019).

Measurement of m 6 A modification
EpiQuik m 6 A RNA Methylation Quantification Kit was used to measure the amounts of m 6 A in total RNA (P-9005; Epigentek Group Inc., Farmingdale, NY, USA).200 ng of RNA were added to the assay wells together with the m 6 A standard, and then capture and detection antibody solutions were added.By measuring each well's absorbance at a wavelength of 450 nm (OD450), the m 6 A levels were then calculated colorimetrically and determined using the standard curve.

Immunoprecipitation
Separated MDA-MB-231 and HEK-293T cells were lysed in protease inhibitor-containing RIPA lysis buffer (Beyotime).We measured protein concentrations using a bicinchoninic acid (BCA) protein assay kit (Beyotime).Protein A/G-agarose beads (Beyotime) were used to preclear cell lysates for 0.5 h.Next, the lysates were immunoprecipitated with the appropriate antibodies overnight at 4 • C. The lysates underwent a second 3-hour incubation with protein A/G-agarose beads the next day.The RIPA lysis solution was used to wash the immunocomplexes five times, after which the bound proteins were boiled to release them and exposed to SDS-PAGE for Western blot analysis.

Immunoprecipitation coupled with mass spectrometry (IP/MS)
Total proteins were extracted from HEK-293T cells and immunoprecipitated as previously mentioned using the proper primary antibody and protein A/G-agarose beads (Beyotime).Mass spectrometry was used to assess the extracted immunoprecipitates.

Immunohistochemical analysis
All samples and tumours that were implanted were fixed in 4% formalin before being paraffin embedded.Sections (thickness, 4 m) were treated with primary antibodies for the specific detection of USP19, BAG6, CHOP, GRP78 or Ki67 (proteintech, China) for an overnight incubation at 4 • C after blocking endogenous peroxides and proteins.Sections were treated with secondary antibodies that were HRP-polymer-conjugated for 1 h at 37 • C. Sections were then stained for 3 min in 3,3-diaminobenzidine solution and hematoxylin was used as a counterstain on the nuclei.Sections of tumour were evaluated in a blinded way.Based on three randomly chosen fields for each section, the percentage of tumours that tested positive and the degree of cell staining were calculated.A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.

Bioinformatic analyses
By using the Bioconductor packages "DESeq2," "limma," and "GSEA," as well as the R packages "DESeq2" and "GSEA", we were able to identify differentially expressed genes (DEGs).We used Gene Set Enrichment Analysis (GSEA) enrichment analysis to look at the distinct RNA modification patterns in biological processes.GSEA software revealed that tumour hallmarks were more prevalent in the high-expression group compared to the low-expression group (normalized enrichment score (NES) > 1, p-value < .05,false discovery rate (FDR) < .25).

TUNEL assay
The In Situ CellDeath Detection Kit was used in a terminal deoxynucleotidyl transferase-mediated dUTP nickend labelling (TUNEL) experiment (Servicebio, China).Dewaxing, rehydrating, incubation and other steps of paraffin slices were done in accordance with procedure.
The sample was treated with three washes of PBS before being treated with a TUNEL reaction mixture containing TdT and dUTP (1:9) and incubated at 37 • C for 2 h in a humid environment.The samples were examined under a fluorescence microscope after being counterstained with DAPI.The formula used to determine the cancer cells' apoptotic index was as follows: apoptotic index = apoptotic cells/total cells 100%.

Study approval
All animal experiments are in accordance with the Public Health Service Policy on Humane Care of Laboratory Animals and approved by Institutional Animal Care and Use Committee (IACUC) of Gempharmatech Co., Ltd (AP#: 2107052).

Statistics analysis
At least three distinct biological replicates are included in the data, which are displayed as mean SD ± SD.The statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software) and R software (version 4.0.1).Prior to doing any parametric studies, we checked the distribution's normality and the equality of the variance.For comparisons between two groups, the unpaired, two-tailed Student's t-test was used, and for comparisons involving more than two groups, the one-or two-way ANOVA was followed by the Tukey's post hoc test (normality and equal variance passed).When normality and/or equal variance failed, the Wilcoxon rank-sum test and the Kruskal-Wallis test were used to analyse nonparametric data.Statistical significance was defined as a p-value less than 0.05.Kaplan-Meier survival curves were also employed to compare the overall survival in two subgroups.

USP19 was down-regulated in triple-negative breast cancer tissues and cells
To gain insight into the functions of ubiquitin modification in BC, we performed a screen interrogating expression data for 84 human DUB genes in TCGA 26 and the Genotype-Tissue Expression (GTEx) databases. 27After conducting an analysis of USP19 expression in BC and adjacent tissues, our findings indicate that there was no significant difference in USP19 expression between cancer and adjacent normal tissues (Figure 1A).Following that, we carried out an independent analysis to examine the expression of USP19 across various subtypes of BC.Our findings revealed that the expression level of USP19 was significantly lower in the basel-like subtypes when compared to paracancer tissues (Figure 1B).Moreover, patients with high USP19 expression exhibited a better prognosis (Figure 1C).The correlation result between the expression of USP19 and the prognosis of BC patients is provided in Figure S2C.We further examined USP19 expression in different subtypes of BC in TCGA dataset.As shown in Figure 1D, USP19 expression was lower in the basal-like subtype than that in other subtypes of BC.This conclusion was confirmed in our centre, by sequencing 196 BC tissue samples (Figure 1E).According to GSEA, the group with high expression of USP19 was more linked with calcium ion transmembrane transport, ubiquitin like protein ligase activity, intramolecular oxidoreductase activity, KEGG apoptosis, PI3K AKT MTOR signalling and UPR (Figure 1F,G).We further examined USP19 expression in normal mammary epithelial cells (MCF-10A) and BC cells lines (MDA-MB-231, MDA-MB-468, BT-549, Hs-578T, MCF-7, SK-BR-3, T47D, and ZR75-1) by qRT-PCR and Western blot.USP19 expression was lower in the TNBC cell lines than that is normal mammary epithelial cells (Figure 1H,I).In order to examine USP19 expression in paraffin-embedded TNBC tissues, an IHC assay was used.TNBC tissues had lower levels of USP19 protein expression when compared to adjacent tissues (Figure 1J).To examine its biological roles in TNBC, MDA-MB-231 and BT-549 cells were selected for transfection with USP19 siRNA and Myc-tagged plasmid constructs.USP19 was significantly regulated in MDA-MB-231 compared with the levels detected in the control groups (Figure S1F).

USP19 suppressed cell proliferation and induced apoptosis of BC cell lines
The colony formation assay was used to investigate the long-term impact of USP19 on cell proliferation.Results revealed that USP19 overexpression impaired colony formation ability (Figure 2A).In addition, the 5-ethynyl-2′deoxyuridine (EdU) incorporation test was used to assess the effect of USP19 on proliferation, in a more sensitive and specific manner.As shown in Figure 2B, the number of TNBC cells incorporating EdU in the USP19-plasmidtreated group was much lower than in the control group.Functionally, Fluo-4 AM staining was used to assess intracellular Ca 2+ levels.Fluorescence microscopy and flow cytometry revealed a substantial rise in intracellular Ca 2+ concentration in USP19-plasmid-transfected cells, indicating that USP19 could enhance Ca 2+ levels (Figure 2C).
The CCK-8 method was then used to assess the effect of USP19 on TNBC cell growth.In comparison to the control group, the proliferation rate of MDA-MB-231 and BT-549 cells transfected with USP19-plasmid was much lower (Figure 2D).The idea that USP19 operates as a potential tumour suppressor gene in TNBC was confirmed by FCM measurement of apoptosis.When compared to the control group, cells overexpressing USP19 had a greater apoptotic rate (Figure 2E).The results of the CCK-8 cell viability assays matched those of the flow cytometry study.Based on these observations, we hypothesised that USP19 promotes BC cell death.Flow cytometry was used to investigate the role of cell-cycle disruption in the increased proliferation caused by USP19 overexpression.We discovered that USP19 overexpression gradually reduced the proportion of cells in the G1 phase and increased the proportion of cells in the G2 and S phases, confirming the induction of cell cycle arrest in the G2 and S phases after USP19 overexpression (Figure 2F,G).BC cells that had been transduced with USP19-targeting plasmid were injected into the flank of 5-weeks old female nude mice to further demonstrate the tumour suppression role of USP19 in vivo (Figure 2H).From the day 5 after injection, we began to calculate the tumor volume every 5 days until tumour sizes approached 1500 mm 3 (day 20).Consistent with in vitro experiments, overexpression of endogenous USP19 markedly attenuated both the tumour size and weight in vivo.The evidence that we stably transfected cell lines for in vivo experiments are provided in the new uploaded Figure S2A.TUNEL staining showed that the proportion of TUNELpositive cells increased in the oeUSP19 (overexpression USP19) group when compared with the control.Tumour samples from each group were stained for Ki67, demonstrating that USP19 overexpression tumour-bearing mice had much lower cell proliferation marker Ki67 (Figure 2I).It was clear that tumour development rates in the USP19 overexpression groups were much slower than those of the control groups, and tumour weights were also significantly reduced.These results unequivocally demonstrated the critical function of USP19 in regulating TNBC cell proliferation.Subsequently, we investigated the impact of the C607S-USP19 mutant and siUSP19-2 on the proliferation and apoptosis of TNBC cells.Figure S1A-S1E presents the experimental findings.

USP19 interacted with and stabilises BAG6
To identify putative interacting proteins for USP19, we constructed human embryonic kidney (HEK) 293T cells stably overexpressing USP19, by transfecting Myc-tagged plasmids which would increase the interaction of proteins with USP19.We investigated which proteins interact with USP19 using immunoprecipitation/mass spectrometry (IP/MS), therefore elucidating the underlying mechanism of USP19 in the regulation of TNBC cell proliferation and apoptosis.Numerous potential USP19-interacting proteins were identified; among these proteins was BAG6 which combines with the tail-anchored protein to recognise the hydrophobicity of the polypeptide synthesised by the ribosome, and promotes its entry into the ER.BAG6 simultaneously enhanced ubiquitin/proteasome-mediated degradation of mis-localised proteins, and was regarded as a putative interacting protein with USP19 (Figure 3A,B).We next researched the effect of knockdown of USP19 on the BAG6 expression levels in the MDA-MB-231 cell line.The endogenous BAG6 protein levels were downregulated in MDA-MB-231 cells after knocking down USP19 (siUSP19-2 was selected for follow-up experiments, Figure 3C).
The reduction in BAG6 protein levels caused by USP19 knockdown was reversed after treatment with proteasome inhibitor MG132.Western blotting was conducted using an anti-ubiquitin antibody, demonstrating that the activity of MG132 functions as a proteasome inhibitor (Figure 3D).Knockdown of USP19 gene did not cause changes in BAG6 protein levels with lysosome inhibitor Chloroquine (CQ) (Figure S3B).We exclude the effect of USP19 on the lysosomal degradation pathway.Confocal imaging showed that the BAG6 fluorescence signal was significantly enhanced after overexpression of USP19, which revealed the colocalization of BAG6 and USP19 (Figure 3E).Silencing and overexpressing USP19 significantly reduced BAG6 protein levels, despite having no effect on BAG6 mRNA levels (Figure 3F and Figure S3A), suggesting that USP19 regulates BAG6 not at the transcript but at the protein translation level.Additionally, the purified GST-USP19 and GST-BAG6 proteins produced in E. coli BL21 were used in an in vitro GST pull-down test.BAG6 was subsequently treated with purified GST-USP19 protein that had been immobilised on protein G beads (Figure 3G).Next, we examined the binding association of BAG6 and USP19 in cells by IP-Western blot analysis.Ectopically expressed BAG6 was readily detected in the USP19 immune complex, and reverse coimmunoprecipitation (Co-IP) confirmed that USP19 could also be detected in the BAG6 immune complex (Figure 3H).We also have conducted an IP/WB assay to showcase the interaction between USP19 and BAG6 in both breast normal and TNBC cell lines (MCF-10A and MDA-MB-231) (Figure 3I,J).We also explored whether BAG6 interacts with USP19 mRNA directly.This hypothesis was negated by RNA pull-down and immunoprecipitation assays (Figure S2B.).We transfected HEK-293T cells with plasmids tagged with Myc and Flag epitopes, and Co-IP experiments confirmed that Myc-tagged USP19 can co-precipitate with Flag-tagged BAG6 (Figure S2D).We investigated the effect of USP19 (up-or down-regulation) on the stability of endogenous BAG6 protein level in the presence of the protein synthesis inhibitor cycloheximide (CHX).Overexpression of USP19 significantly inhibited the degradation of BAG6, whereas knockdown of USP19 markedly promoted BAG6 degradation (Figure 3K-3M and Figure S2E).

USP19 removed the K29-linked ubiquitin chains of BAG6
We also investigated whether USP19 acts as a deubiquitination enzyme to stabilise BAG6 expression.We used immunoblot analysis with an anti-Flag antibody to analyse the function of USP19 as deubiquitinase of BAG6, followed by immunoprecipitation with anti-HA for total cell ubiquitin.We found that USP19 regulates the BAG6 level through deubiquitination modification.Overexpression of USP19 significantly decreased ubiquitination of BAG6, but increased BAG6 protein level when compared with the control group (Figure 4A).To further clarify the regulatory role of BAG6 ubiquitination by USP19, MDA-MB-231 cells were co-transfected with Myc-USP19 (wild-type, WT) or Myc-C607S mutant, 10 Flag-BAG6, and HA-Ub.Next, to investigate whether the ability of USP19 to deubiquitinate BAG6 was dependent on its protease activity, we transfected WT-USP19 or its mutants into cells.Overexpression of WT-USP19 reduced BAG6 ubiquitination, but F I G U R E 4 USP19 inhibits BAG6 ubiquitination.(A) HEK-293T cells were transfected with Myc-tagged USP19, Flag-BAG6, HA-UB or Vector and protein extracts were harvested after MG132 (10 μM) treatment for 3 h.Protein extracts were immunoprecipitated using anti-flag antibody and analysed by immunoblot using anti-HA and, anti-Myc, anti-flag and anti-α-Tubulin antibodies.(B) Lysates from HEK-293T cells transfected with Myc-tagged USP19 (WT) or Myc-tagged USP19 (C607S), together with HA-tagged Ub and Flag-tagged BAG6, were immunoprecipitated with anti-flag and immunoblotted with anti-HA, anti-Myc and anti-flag.(C) Lysates of HEK-293T cells transfected with plasmid expressing Flag-BAG6 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub, or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h, were immunoprecipitated with anti-flag and immunoblotted with anti-HA.D. siUSP19, a plasmid expressing flag-BAG6, and K29-linked-Ub were transfected into HEK-293T cells.Protein was extracted after being exposed to MG132 (10 μM) for 3 h, and it was then immunoprecipitated with anti-flag and immunoblotted with anti-HA.A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.transfection with the C607S-USP19 plasmid had no effect on BAG6 ubiquitination (Figure 4B).Then, we investigated which types of BAG6 ubiquitination are regulated by USP19.Different processes of polyubiquitination, including ubiquitination of lysine 6 (K6)-, K11-, K27-, K29-, K33-, K48 and K63-linked diubiquitin, have been implicated in the regulation of intracellular protein levels. 28,29Overexpression of USP19 significantly inhibited the WT and K29-linked polyubiquitination of BAG6, but did not affect the other ubiquitination linkages (K6, K11, K27, K33, K48 or K63) in BAG6 (Figure 4C).This was in agreement with USP19 knockdown increasing K29-linked ubiquitination of BAG6 (Figure 4D).In summary, these results demonstrated that the K29 linkage was essential for USP19 to exert its deubiquitination function and modify BAG6.

BAG6 inhibition suppressed USP19 overexpression-induced events
We demonstrated that expression of USP19 inhibited proliferation and induced apoptosis in TNBC cells, as well as promoting BAG6 protein expression by deubiquitinating modification.To further verify whether the effects of USP19 on proliferation in TNBC cells were mediated by regulation of BAG6, we knockdowned BAG6 in MDA-MB-231 and BT-549 cells (Figure 5A-C).Besides, we supplemented the experiments on the effect of BAG6 overexpression on proliferation in cells and tumour growth in Figure 3C,D.Subsequently, we investigated the ability of BAG6 to counteract the effects of USP19 upregulation.BC cells were co-transfected with USP19 plasmid and BAG6 siRNA.As shown in flow cytometry (Figure 5D-F), knock-down of endogenous BAG6 inhibited cell apoptosis, while attenuating cell cycle arrest in the G2 and S phases, which was opposite of the effects of USP19 overexpression.The results showed that BAG6 knockdown effectively reversed the promotion of proliferation and suppression of apoptosis induced by USP19 overexpression (Figure 5A-F).The above results confirmed our hypothesis, namely that USP19 directly targets BAG6 to inhibit BC cell proliferation and induce apoptosis.
To clarify the role of USP19 in cancer cell suppression through regulation of BAG6 in vivo, MDA-MB-231 cells with expressed oeUSP19, oeUSP19/siBAG6, and control plasmids (Figure 5F), were subcutaneously injected into the right armpits of BALB/c nude mice (6 mice per group).The evidence that we stably transfected cell lines for in vivo experiments are provided in the new uploaded Figure S2A.From the day 5 after injection, we began to calculate the tumour volume every 5 days until tumour sizes approached 1500 mm 3 (day 20).Consistent with in vitro experiments, overexpression of endogenous BAG6 markedly attenuated both the tumour size and weight in vivo.TUNEL staining showed that the proportion of positive cells increased in the oeUSP19 group when compared with the control (Figure 5H).Tumour samples from each group were stained for Ki67, demonstrating that tumour-bearing mice had much more up-regulation of the siBAG6-induced cell proliferation marker Ki67 (Figure 5H).From rescue experiments, we also found that the effect of USP19 could be compensated with BAG6 expression.

BAG6 targeted BCL2 proteins to increase intracellular calcium
Next, we further searched for the specific mechanism by which BAG6 regulates TNBC progression.By transfecting flag-tagged plasmids, which would enhance the interaction of proteins with BAG6, we created HEK-293T cells that could consistently overexpress BAG6.By utilising IP-MS, we looked at the proteins that interact with BAG6 in order to clarify the underlying mechanism of BAG6 controls the course of TNBC (Figure 6A).His-BCL2 interacted with Flag-BAG6 according to Co-IP and immunoblot analyses.The binding relationship between BCL2 and BAG6 in cells was then investigated using an IP-Western blot analysis.The BAG6 immune complex was easily able to identify ectopically produced BCL2, and reverse Co-IP indicated that BAG6 may also be found in the BCL2 immune complex (Figure 6B).The findings showed that BAG6 overexpression significantly shortened the Bcl-2 protein's half-life in MDA-MB-231 cells (Figure 6C).The impact of USP19 and BAG6 on BCL2 expression was then investigated on our further research.After overexpressing USP19 and BAG6, the endogenous BCL2 protein levels were reduced in MDA-MB-231 cells (Figure 6D).In MDA-MB-231 cells, Co-IP with epitope-tagged BAG6 and BCL2 was also carried out.Co-transfection with Flag-BAG6 and His-BCL2 expressing plasmids was carried out in MDA-MB-231 cells to further demonstrate the link between BAG6 and BCL2.Results indicated that exogenous BCL2 might interact with ectopic BAG6 expression (Figure 6E).BAG6 is identified as a ubiquitin-like protein 30 that shut-tles between the cytoplasm and nucleus.It was also involved in the regulation of apoptosis, antigen presentation and T-cell response. 30,31Furthermore, BAG6 could suppress RNA virus-mediated innate immunity by increasing VISA/TRAF2 K48 poly-ubiquitination. 32 Clearly, the upregulation of BAG6 greatly promoted BCL2 ubiquitination in MDA-MB-231 cells (Figure 6F).A GST pull-down assay in vitro was performed using the purified GST-BAG6 and GST-BCL2 proteins generated in E. coli BL21.Following that, BCL2 was treated with pure GST-BAG6 protein that had been immobilised on protein G beads (Figure 6G).It has been observed that BCL2 can control the activity of the intracellular Ca 2+ channel inositol 1,4,5trisphosphate receptor (IP3R) and the release of calcium during apoptosis. 33ER stress, which compromises appropriate protein folding and intracellular transport, may be brought on by calcium homeostasis.The impact of BCL2 knockdown on IP3R and p-IP3R expression levels in the MDA-MB-231 cell line was the subject of our subsequent investigation.After BCL2 knocking down, the endogenous p-IP3R protein levels in MDA-MB-231 cells were reduced.But there was no substantial change in the IP3R expression (Figure 6H).Fluorescence microscopy and flow cytometry revealed a substantial rise in intracellular Ca 2+ concentration in USP19 plasmid and siBAG6 transfected cells, indicating that USP19/BAG6 axis could enhance Ca 2+ levels through inhibiting BCL2 expression (Figure 6I,J).Fluorescence microscopy revealed a substantial rise in intracellular Ca 2+ concentration in BCL2 plasmid transfected cells, indicating that BCL2 could enhance Ca 2+ levels in Figure S3E.
To further clarify the regulatory role of BCL2 ubiquitination by USP19 and BAG6, MDA-MB-231 cells were cotransfected with Myc-USP19, siUSP19, Flag-BAG6, siBAG6 and HA-Ub.According to the aforementioned findings, USP19 stabilises BAG6 protein, which in turn impacts ubiquitination of BCL2, but it has no effect on BCL2 protein's intrinsic ubiquitin level (Figure 6K).The expression level of USP19 remained unchanged after overexpressing or knocking down the BAG6 protein, indicating that BAG6 had no impact on the protein level of USP19 (Figure 6L).

USP19 promoted apoptosis through BAG6-induced ER stress in human BC cells
According to gene set enrichment analysis, high expression of USP19 appeared to be more correlated with the UPR (Figure 7A).BAG6, an abundant cytoplasmic chaperone, is related to the biogenesis of ER tail-anchored membrane proteins, to the quality control of ribosomedeficient plastids and mislocalised proteins, and acts to increase the efficiency of the ER-associated protein degra-F I G U R E 6 BAG6 interacts with and degrades BCL2.(A) IP/MS analysis indicated that BCL2 is an interacting protein that binds to BAG6.(B) Endogenous protein interactions were confirmed in MDA-MB-231 lysates by immunoprecipitation with anti-BAG6, followed by immunoblotting with anti-BCL2 or BAG6, respectively.(C) BCL2 protein levels in Vector and Flag-BAG6 MDA-MB-231 were evaluated by immunoblotting with anti-BCL2 and anti-BAG6 in the presence of cycloheximide (CHX, 10 μg/mL) for indicated timepoint.(D) The impact of USP19 and BAG6 on BCL2 expression.(E)) Exogenous protein interactions were confirmed in MDA-MB-231 lysates by immunoprecipitation with anti-BAG6, followed by immunoblotting with anti-BCL2 or BAG6, respectively.(F) MDA-MB-231 cells were transfected with Flag-tagged BAG6, His-BCL2, HA-UB or Vector and protein extracts were harvested after MG132 (10 μM) treatment for 3 h.Protein extracts were immunoprecipitated using anti-His antibody and analysed by immunoblot using anti-HA and, anti-Flag, anti-His and anti-α-Tubulin antibodies.(G) The interaction between BAG6 and BCL2 was assessed using a GST pull-down assay.All proteins were detected using the indicated antibodies.(H) Changes in endogenous IP3R and p-IP3R expression following siBCL2 transfection.(I) Fluorescence microscopy was used to observe Fluo-4 AM-loaded MDA-MB-231 and BT549 cells.Fluo-4 fluorescence (green) increases with intracellular Ca 2+ concentration.(J) Intracellular Ca 2+ levels in the various groups were revealed by flow cytometry analysis.(K) MDA-MB-231 cells were transfected with Myc-tagged USP19, siUSP19, Flag-BAG6, siBAG6, HA-UB or Vector and protein extracts were harvested after MG132 (10 μM) treatment for 3 h.Protein extracts were immunoprecipitated using anti-BCL2 antibody and analysed by immunoblot using anti-HA and, anti-BAG6, anti-BCL2 and anti-α-Tubulin antibodies.(L) The impact of BAG6 on USP19 expression.A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.dation (ERAD) system.We investigated whether USP19 influences TNBC development via BAG6-mediated regulation of ER stress.To further investigate the effect of USP19 on BAG6-induced ERAD, we validated the expression levels of ATF4, CHOP, p-IRE, GRP78 and XBP1 in animal models by western blotting (Figure 7B-7D).Histological and immunohistochemical (IHC) analyses found that USP19 enhanced the expression of ER stress-related proteins to induce the ER stress response (Figure 7E).Taken together, these results indicated that USP19 can induce the activation of ER stress-related apoptosis through stabilization of BAG6 expression levels, thereby ameliorating TNBC progression in vivo.

METTL14 reduced m 6 A RNA methylation of USP19
Lastly, after exploring the function of USP19 in BC cells, we wondered why the expression of USP19 was decreased in TBNC.N 6 -methyladenosine (m 6 A) modification is the most classic intracellular mRNA post-transcriptional modification and is involved in the regulation of various biological processes.We speculated that the m 6 A modification might be responsible for the downregulation of USP19.To test this hypothesis, we examined METTL14 mRNA levels in TNBC tissues by qRT-PCR, and discovered that the expression of METTL14 was significantly upregulated in TNBC tissues (Figure 7F).A significant increase in the m 6 A level in mRNAs was also found in TNBC tissues (Figure 7F).Moreover, the USP19 mRNA level was significantly down-regulated after overexpression of METTL14 (Figure 7G), and overexpression of METTL14 markedly decreased the half-life of USP19 mRNA in MDA-MB-231 cells (Figure 7G).We treated MDA-MB-231 cells with cycloleucine, which has been extensively utilised in RNA methylation studies and might lower mRNA methylation.Cycloleucine was able to decrease the expression of USP19 without affecting METTL14 (Figure 5E,F).Collectively, the above results clarified that METTL14mediated m 6 A modification results for the downregulation of USP19 mRNA levels.We treated MDA-MB-231 cells with cycloleucine, which has been extensively utilised in RNA methylation studies and might lower mRNA methylation.(Figure S3F).

DISCUSSION
Estrogen receptor, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are lost in TNBC, which accounts for approximately 10%-20% of BCs. 34However, TNBC tended to be poorly differentiated and more prone to recurrence and metastasis than other BC subtypes.TNBC with recurrence and metastasis was more aggressive and less sensitive to radiotherapy and chemotherapy.Patients with advanced TNBC had the shortest median overall survival of approximately 18 months (13.6-20.1 months). 35Therefore, it was very important to clarify the pathogenesis and factors inducing TNBC.
Using both in vitro and in vivo assays, we first showed that USP19 was significantly downregulated in TNBC and that overexpression impaired colony formation ability in TNBC cell lines.USP19 was shown to inhibit cell proliferation, while inducing apoptosis by reducing ubiquitination and degradation of BAG6 and enhancing BCL2 induced ER stress by elevating the intracellular Ca 2+ concentration.Therefore, we identified USP19 as a potential target for TNBC therapy and elucidated the possible mechanism by which USP19 inhibits TNBC progression.7][38] It has been reported to be critical for the regulation of programmed cell death. 39,40 recent publication showed that HOIL1 E3 mediates mono-ubiquitination of the linear ubiquitin chain assembly complex, resulting in embryonic death upon knockout of HOIL1 in mice.This process can be achieved by knockout of caspase-8 and rescued by MLKL, suggesting the distinct interaction between cell death and ubiquitination. 41A study found that PELI1 can promote K48-linked ubiquitination of RIP3 on Lys363, leading to proteasomal degradation, thereby inhibiting necroptosis. 42t was also demonstrated that CYLD promotes necroptosis after deubiquitination of RIP1 in necrosomes. 38,43 stabilises its expression, thereby maintaining the DNA damage response in BC cells. 44Sun found that USP36 deubiquitinates c-Myc in the nucleolus and acts downstream of c-Myc to promote BC proliferation. 45AKT was found to promote nuclear export after phosphorylation of USP4 and removes TβRI ubiquitination, promoting TGFβ-induced epithelial-to-mesenchymal transition in BC. 46 Deubiquitination thus plays a crucial role in the development of BC.In this study, we found, for the first time, that USP19 reduced ubiquitination and degradation of BAG6 to induce BC cell apoptosis and ER stress.USP19 was reported to interact with TRIF and remove the K27-linked polyubiquitin moiety of TRIF, thereby inhibiting TRIF recruitment to TLR3/4 and promoting innate immune responses. 47Nevertheless, the functional role of USP19 in cell biology, particularly in the regulation of ER stress, was still unclear.
It is reported that seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) can be attached to the Homotypic Ub polymer's C-terminus. 48Usually, K48-linked ubiquitin chains are responsible for target proteins' degradation.However, other ubiquitin chains also play important roles in the cellular function.Deubiquitinase (DUB) TRABID was shown to preferentially hydrolyse K29 and K33 links. 49u et al. revealed that UBE3C and TRABID, two ubiquitin ligases, cooperate reciprocally to regulate VPS34 through K29/K48-branched ubiquitin chains. 50TRIP12 selectively assembles K29-linked ubiquitin chains, boosting the development of K29/K48 branched ubiquitination. 51In this paper, we found that USP19 removed the K29-linked ubiquitin chains of BAG6 and stabilised the latter's protein level.
ER stress is a highly dynamic process that may lead to abnormal apoptosis and death.Genetic alterations can also promote ER stress and persistent activation of the UPR in tumour cells.Excessive activation of tumour protooncogenes could lead to increased protein synthesis, while proliferating cells require rapid ER amplification to divide into daughter cells. 52In addition, multiple factors, such as chemotherapy, induce a lethal ER stress response with immunogenic cell death triggers, which activates a protective antitumor immune response. 15Here, we showed that USP19 binds to BAG6 and decreases BAG6 ubiquitination, which prevents BAG6 from being degraded in TNBC tumour tissue and BC cells.Knockdown of BAG6 in mice also reversed ER stress triggered by USP19, indicating that the essential effects of USP19 on ER stress depend on BAG6 stability.Kawahara et al. 30,53 identified BAG6 as a cytosolic interactor of misfolded proteins from ER by MS analysis, showing that BAG6 specifically and abundantly bound to the deglycosylated version of the misfolded protein.Misfolded protein is known to be a key indicator of ER stress.These findings suggested that USP19 controls ER stress, most likely through controlling BAG6 stability.Although the majority of our recent work has been focused on TNBC cell lines, USP19 overexpression mice have allowed us to more thoroughly study and confirm the specific functional role of USP19.Therefore, we next investigated the molecular mechanism by which USP19 induces changes in the ER stress by affecting the expression of BAG6.
5][56] It is one of the most significant controllers of apoptosis.The BCL2 family is often classified into two groups: pro-survival BCL2 proteins (such as BCL2, A1, Bcl-xL and Mcl1) and proapoptotic BCL2 proteins (such as Bad, Bax/Bak, Bim, Bik and Puma). 57,58Signals coming from or focusing on intracellular organelles, such as the ER or the mitochondria, are frequently responsible for controlling the apoptosis process. 59Growing data suggest that the BCL2 family regulates apoptosis in the ER, the major Ca 2+ cellular storage organelle.BAG6 is a protein that resembles ubiquitin, according to Kawahara.In this paper, we showed that BAG6 binds to BCL2 and increases BCL2 ubiquitination, which decrease BCL2 accumulation in BC cells.Cytotoxic stress and cell death may be brought on by cellular Ca 2+ excess or disruptions of intracellular Ca 2+ storage. 33IP3R is a calcium channel that resides in the ER.Each subunit of the tetrameric IP3R protein comprises a transmembrane pore region at C-terminus, a ligand binding domain at N-terminus and an intervening modulatory domain. 60Numerous cellular activities, including metabolism, gene expression and apoptosis, are regulated by calcium release via the IP3R. 61,62BCL2 overexpression has been demonstrated to lower ER Ca 2+ storage and inhibit capacitative Ca 2+ entry following ER Ca 2+ release. 63,64It was found that BCL2's BH4 domain facilitated a connection with intracellular Ca 2+ channel the inositol 1,4,5-trisphosphate receptor (IP3R) to control cell growth and survival. 33,60,65,66In MDA-MB-231 cells, endogenous p-IP3R protein levels were decreased when BCL2 was knocked down, while IP3R expression remained mostly unchanged.We discovered that ubiquitin-like protein BAG6 can elevate the intracellular Ca 2+ concentration to alter ER stress via regulating the level of BCL2 ubiquitination and decreasing IP3R phosphorylation modification level.Our findings implied that USP19 can be a possible therapeutic target in BC, together with evidence revealing that decreased expression of USP19 indicated a poorer prognosis in this disease.
Multiple perturbations to cells can lead to accumulation of unfolded proteins in the ER, which triggers the UPR when UPs accumulate to a certain amount. 67,68When the primary stimuli that cause protein unfolding in the ER are prolonged or excessive, the adaptive mechanisms initiated by the UPR fail to compensate, and cell death by apoptosis will be triggered. 69Activated IRE1α promotes the expression of XBP1 and other canonical UPR genes, such as foldase, chaperone, and ERAD machinery.IRE1α-XBP1 could regulate the degradation of misfolded proteins and enhance the folding and secretion of proteins to play an important role in the occurrence of the UPR.1][72] Our results revealed that the levels of GRP78, ATF4, CHOP, p-IRE, and XBP1 expression were increased in mouse tumour tissue overexpressing USP19, indicating an increased ER stress parallel to the expression of USP19 in TNBC tumorigenesis.ER stress-related proteins were also significantly up-regulated in human BC cells overexpressing USP19 compared with normal controls.Collectively, USP19 can significantly increase the unfolded protein entering the ER and induce ER stress, by reducing BAG6 ubiquitination to kill TNBC cells.m 6 A methylation had received a lot of attention recently and was now widely acknowledged as the most frequent and prolific alteration on mRNA and as a mechanism to control RNA translation, stability, and degradation. 73,74We found that total m 6 A levels were upregulated in TNBC tissues, along with elevated m 6 A "writer" METTL14 levels.Sun et al. 75 reported that LNC942 drew METTL14 protein directly due to its unique recognition sequence (+176 to +265).By modifying post-transcriptional m 6 A, this stabilises the downstream expression of LNC942, including CXCR4 and CYP1B1.To determine whether m 6 A modification was associated with the regulation of USP19 mRNA, we overexpressed METTL14 in MDA-MB-231, and found that the expression of USP19 mRNA was decreased when METTL14 was overexpressed.RNA stability experiments demonstrated that the stability of USP19 mRNA was correspondingly decreased when METTL14 was overexpressed.In an initial investigation of the upstream regulatory mechanism of USP19, we discovered that m 6 A modification of USP19 resulted in USP19 downregulation.Further detailed investigations are needed in subsequent studies to fully confirm this conclusion.
In conclusion, this study showed that USP19 played a key role in TNBC cancer proliferation and apoptosis.The pace of mouse tumour development was considerably reduced by targeting USP19.Additionally, our research identified a novel mechanism by which USP19 elevates intracellular Ca 2+ levels to induce ER stress in TNBC, through regulating the ubiquitination and degradation of BAG6 and BCL2.Additionally, we demonstrated that USP19 was downregulated at the mRNA level, as a result of increased m 6 A alteration.The bioinformatics analysis and our patient data, in particular, improved the relevance and dependability of the findings on USP19 and its associated pathways.Since only deubiquitinases were examined in this investigation, it was possible that additional functional proteins may play a role in the control of ER stress and apoptosis in TNBC mice.Our findings indicated that targeting USP19 /BAG6/BCL2 axis may be an efficient therapeutic target for treating TNBC.

A C K N O W L E D G M E N T S
The current study was funded by the National Natural Science Foundation of China (Grant Nos.82072931 and 82002805) and Jiangsu Province Capability Improvement Project through Science, Technology and Education (Jiangsu Provincial Medical Key Discipline, ZDXK202222).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Supporting information accompanies this paper at Supplementary Tables and Materials.

F I G U R E 1
Expression of USP19 in BC tissues, BC cells and transfected cells.(A,B) The expression of USP19 in different tissues (Basal-like, BC (breast cancer) and normal tissues) from TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) cohort.(C) Kaplan-Meier curves showed the stratification analysis of the USP19 in TNBC tissues.(D) The expression level of USP19 in subtypes of BC in TCGA cohort.(E) The expression level of USP19 in subtypes of BC in TCGA cohort and 196 breast cancer tissue samples RNA sequencing data.(F,G) Gene set enrichment analysis (GSEA) based on the sequencing data of 196 cases showed that calcium ion transmembrane transport, ubiquitin like protein ligase activity, intramolecular oxidoreductase activity, KEGG apoptosis, PI3K AKT MTOR signalling and unfolded protein response was enriched in the USP19 high-expression subgroup.(H,I) The expression levels of USP19 in BC cells and MCF-10A were detected by mRNA qRT-PCR and Western blot.(J) Immunohistochemical staining against USP19 collected from TNBC and adjacent tissue.A representative dataset is displayed as mean ± SEM values.ns.,Not significant, *p < .05,**p < .01,***p < .001.

F
I G U R E 2 USP19 inhibits cell proliferation and induces cell apoptosis.(A) Effects of USP19 expression on the colony formation of BC cells.(B) Representative profiles of EdU cell growth in MDA-MB-231 cells and BT-549 cells after transfection with Myc-USP19 plasmid respectively compared with the control.(C) Fluorescence microscopy was used to observe Fluo-4 AM-loaded MDA-MB-231 and BT549 cells.Fluo-4 fluorescence (green) increases with intracellular Ca 2+ concentration.(D) CCK-8 was used to determine the proliferation of BC cells transfected with Myc-USP19 plasmid.OD value between Myc-USP19 plasmid and corresponding control group was significantly different.The data expressed as the mean ± SD. (E) Evaluation of the impact of altered USP19 expression on cell apoptosis.(F,G) Effects of USP19 modification on BC cell-cycle distribution.(H) Photographs of tumours obtained from the different groups of nude mice transfected with Myc-USP19 plasmid, respectively.The curve graph exhibited the tumour growth measured at different time-points after inoculation (n = 6).The image shows the tumour weight for each group (n = 6).(I) The TUNEL staining in tumor sections and IHC of Ki67 in tumour sections (n = 3 for each group).Statistical results are quantified by ImageJ software.A representative dataset is displayed as mean ± SEM values.ns.,Not significant, *p < .05,**p < .01,***p < .001.

F
I G U R E 3 USP19 interacts with and stabilises BAG6.(A) Coimmunoprecipitation and Coomassie brilliant blue staining analyses of USP19 associated proteins in HEK-293T cells.(B) IP/MS analysis indicated that BAG6 is an interacting protein that binds to USP19.(C) Changes in endogenous BAG6 expression following siUSP19 transfection.(D) Analysis of BAG6, USP19 protein and ubiquitination levels by Western blot in HEK-293T transfected with siUSP19, either with or without proteasome inhibitor MG132.(E) HEK-293T cells were incubated in the DMEM medium for 12 h after transfection Myc-USP19 plasmid for 48 h.Cells were imaged for Cy3-Alexafluor-488 co-localisation.(F) BAG6 mRNA level in HEK-293T transfected with control siRNA and siUSP19.(G) Endogenous protein interactions were confirmed in HEK-293T lysates by immunoprecipitation with anti-USP19, followed by immunoblotting with anti-BAG6 or USP19, respectively.The interaction between USP19 and BAG6 was assessed using a GST pull-down assay.All proteins were detected using the indicated antibodies.(H-J) The binding association of BAG6 and USP19 in cells by IP-Western blot analysis in HEK-293T, MCF-10A and MDA-MB-231.(K-M) BAG6 protein levels in Vector and Myc-USP19 HEK-293T and MDA-MB-231 were evaluated by immunoblotting with anti-BAG6 and anti-USP19 in the presence of cycloheximide (CHX, 10 μg/mL) for indicated timepoint.BAG6 protein levels in control siRNA and USP19 siRNA MDA-MB-231 were evaluated by immunoblotting with anti-BAG6 and anti-USP19 in the presence of CHX (10 μg/mL) for indicated timepoint.A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.

F I G U R E 5
The roles of USP19 and BAG6 in the regulation of breast cancer cell proliferation and apoptosis.(A,B) USP19 up-regulation inhibits proliferation and induces apoptosis using the colony formation assay and EdU assay.The rescue experiments for BAG6 knockdown were performed by USP19 overexpression.(C) Similar rescue experiments for BAG6 silencing were performed in CCK-8 cell viability assays.(D-F) Cell viability assay, flow cytometry and the cell-cycle test are used to demonstrate that USP19 up-regulation triggers apoptosis and reduces proliferation.By overexpressing USP19, BAG6 knockdown rescue studies were carried out.(G) Photographs of mice tumours of each group (n = 6) at the end of the experiment.The curve graph exhibited the tumour growth measured at different time-points after inoculation (n = 6).The image shows the tumour weight for each group (n = 6).(H) The TUNEL staining in tumour sections and IHC of Ki67 in tumour sections (n = 3 for each group).A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.
USP37 can regulate BLM RecQ like helicase deubiquitination and F I G U R E 7 USP19 induced BC apoptosis via endoplasmic reticulum stress (ERS).(A) Based on the sequencing data of 196 cases, gene set enrichment analysis (GSEA) revealed that UNFOLDED PROTEIN RESPONSE was enriched in the USP19 high-expression subgroup.(B,C) WB analysis of GRP78, ATF4, CHOP, p-IRE and XBP1 in mouse tissue with USP19 overexpression and knockdown.(D) WB analysis of GRP78, ATF4, CHOP, p-IRE and XBP1 in MDA-MB-231 and BT-549 with USP19 overexpression and BAG6 knockdown.(E) Representative images of IHC staining of USP19, BAG6, BCL2 and GRP78 in human BC specimens.The TUNEL staining in tumour sections.(F) Relative METTL14 levels in different tissues (Basal-like and normal tissues); m 6 A levels in mRNAs of TNBC and paracancerous tissue.(G) Relative USP19 mRNA levels in MDA-MB-231 transfected with vector or oeMETTL14; RNA lifetime of USP19 in indicated MDA-MB-231 cells as determined by monitoring transcript abundance after transcription inhibition (actinomycin D, 5 μg/mL).H. Working model for regulation of BAG6 stability and endoplasmic reticulum stress by USP19.A representative dataset is displayed as mean ± SEM values.ns., not significant, *p < .05,**p < .01,***p < .001.